Skin flaps inhibit both the current of injury at the amputation surface and regeneration of that limb in newts

For over two decades, we have been investigating a strong (ca. 20-100 microA/cm2), outwardly directed electric current driven through the limb stump for the first few days following amputation in regenerating salamanders. This current is driven through the stump in a proximal/distal direction by the amiloride-sensitive transcutaneous voltage of the intact skin of the stump. Limb regeneration can be manipulated by several technique that manipulate this physiology, demonstrating that the ionic current is necessary, but not sufficient, for normal regeneration of the amphibian limb. Here, we demonstrate that a full thickness graft of skin covering the forelimb stump of newts strikingly inhibits the regeneration of the limb, and that this procedure is also highly correlated to a suppression of peak outwardly directed stump currents in those animals that fail to regenerate.     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

Vasculature in pre-blastema and nerve-dependent blastema stages of regenerating forelimbs of the adult newt, Notophthalmus viridescens

Immunocytochemistry utilizing a monoclonal antibody (BV1; blood vessel 1) highly reactive to the vasculature of the adult newt showed that a developing vasculature was present during early, pre-blastema, and early-bud blastema stages of forelimb regeneration in this species. Infusion of Prussian Blue and DiI into the brachial artery further delineated the intactness of this early vasculature. Finally, macroscopic observations of vascular flow underneath the apical epithelial cap (AEC) and microsurgical removal of the AEC and observation of subsequent bleeding buttressed the conclusion that an intact vasculature exists during early nerve-dependent stages of newt forelimb regeneration. The results suggest that this process of neovascular formation is angiogenesis, i.e., the formation of new vessels from pre-existing vessels in the stump. Furthermore, angiogenesis is an ongoing process initiated early after amputation. Blastema cells and the AEC are likely sourcesof factors that stimulate neovascularization.     

Electrical responses to amputation of the eye in the mystery snail

Immediately following amputation through the eyestalk of the mystery snail (Pomacea), a persistent ionic current enters the apical amputation surface of the eyestalk stump. The circuit is completed by current driven from undamaged integument of the eyestalk stump and other body regions. The current is relatively steady during the first 10 hours following amputation. Currents subsequently begin a slow decline to base line levels by 60 hours postamputation--a time coincident with wound healing processes. The "battery" driving this ionic current is the internally negative transepidermal potential existing across the snail integument--perhaps the result of a net inward pumping of chloride across the skin. This system is compared to other regeneration models such as the amphibian limb, bone fracture repair, and skin wound healing. We suggest that ionic current may be a control of eye regeneration in the snail.     

Blastema cell proliferation in vitro: effects of limb amputation on the mitogenic activity of spinal cord extracts

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19: 7679-7697, 1999, Neuron 28: 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19 : 7679-7697, 1999, Neuron 28 : 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus

An analysis was made of the regeneration of legs and antennae of Oncopeltus. Amputations were performed on first instar larvae within 24 hr after hatching, and on later instars within 24 hr after ecdysis. The resulting regenerates were then measured at each instar. When amputations were performed soon after hatching, there was no significant effect on the duration of any instar. The regenerate was usually visible after the second post-operative ecdysis, and was smaller than a normal appendage (hypomorphic). Removal of the three distal segments of the antenna usually resulted in regeneration of only one segment which was abnormally long and showed a combination of the bristle patterns characteristic of the two most distal segments of the control. In a few such cases a partial intersegmental membrane was present in the regenerated segment. Removal of the tarsus resulted in a structurally complete regenerate which was smaller than the control tarsus. The largest leg regenerates were obtained when amputation was performed through the tibia. With amputation through the femur, a decrease in length of the remainder of this segment was observed after the first ecdysis. This type of amputation and amputation through the trochanter in some cases resulted in the formation of a globular stump containing tarsal claws. The results indicate that amputation of part of an appendage in Oncopeltus does not stimulate an increased growth rate in the stump, but merely causes reorganization of the stump material which subsequently grows at the normal rate. Since even the most hypomorphic regenerates contained well-formed claws, even though proximal parts were missing, it appears that the reorganization process must begin at the most distal point and proceed proximally.     

Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb

A set of tendons, aponeurotic sheets and retinaculae, which transduce muscle action from proximal limb levels to flexion and extension of the digits, is found in limbs of many vertebrates. This set of structures, here termed the digit tendon complex, is described for the axolotl forelimb. We show that the complex forms autonomously in muscleless axolotl limb regenerates produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a year after amputation. The pattern of other connective tissue structures, including the skeleton, is also normal. Fibroblast condensations that may represent sets of these cells normally associated with muscles in the extensor and flexor compartments of the carpal region also form in muscleless limbs. The results are discussed in terms of the importance of the dermis in pattern regulation, selforganization of connective tissues in general and autonomous development of the digit tendon complex in particular.     

Ultrastructural features of the process of wound healing after tail and limb amputation in lizard

Alibardi, L. 2010. Ultrastructural features of the process of wound healing after tail and limb amputation in lizard.—Acta Zoologica (Stockholm)  91 : 306–318 Wound healing and re‐epitelization after amputation of tail and limb in lizard have been studied by electron microscopy to understand the cytological base of immunity to infection in this species. After 2 days post‐amputation in both limb and tail stumps, numerous granulocytes are accumulated over the stump, and participate to the formation of the scab. Bacteria remain confined to the scab or are engulfed by leukocytes and migrating keratinocytes located underneath the scab. Bacteria are degraded within lysosomes present in these cells and are not observed among mesenchymal cells or in blood vessels of the regenerative blastema. Granulocytes, migrating keratinocytes, and later macrophages form an effective barrier responsible for limiting microbe penetration. The innate immunity in lizard is very effective in natural (dirty) condition and impedes the spreading of infection to inner tissues. While the complete re‐epitelization of the tail stump underneath the scab requires 4–7 days, the same process in the limb requires 8–18 or more days post‐amputation, depending from the level of amputation and the persistence of a protruding humerus or femurs on the stump surface. This delay produces the permanence of inflammatory cells such as granulocytes and macrophages in the limb stump for a much longer period than in the tail stump, a process that stimulates scarring.     

Relationship between innervation and forelimb regenerative capacity in the postmetamorphic pond frog Rana brevipoda porosa.

The limb regenerative capacity and the quantity of innervation (the percentage of a cross-sectional area of amputation forelimb stump occupied by nerves) in the pond frog, Rana brevipoda porosa, was investigated in postmetamorphic froglets and adults of various sizes by means of amputating forelimbs through the zeugopodium. Nearly all the amputated limbs of newly metamorphosed froglets, 18-19 mm in snout-vent length, showed heteromorphic regeneration. However, the larger the body size, the lower the presence of limb regeneration. Limb regenerative capacity was completely lost in froglets and adults with snout-vents larger than 35 mm. The quantity of innervation of limbs was highest in newly metamorphosed froglets, gradually decreasing with growth. The nerve quantity in adults with a snout-vent length between 60-67 mm was approximately half that of the froglets. When the nerve supply was augmented by deviating ipsilateral sciatic nerve bundles to the forelimb stump, almost all limbs, which were usually non-regenerative with normal innervation, regenerated heteromorphically. These results show that the decline in limb regenerative capacity during postmetamorphic growth is in part attributable to the reduction in innervation levels to below the threshold level required for regeneration.     

Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axolotl forelimbs

Rotation of skin cuffs 180° around the longitudinal axis of the underlying tissues in the axolotl forelimb results in a high percentage of multiple regenerates after amputation through the rotated skin. Similar results occur after rotation of only the anteroposterior (A-P) axis of the skin. Rotation of only the proximodistal (Pr-Ds) axis of the skin results in normal regenerates whereas dorsoventral (D-V) axial skin rotation results in single regenerates with some disturbances in symmetry. Rotation of anterior or posterior half cuffs of skin produces results similar to those obtained after A-P rotation of full skin cuffs, and rotation of dorsal or ventral skin halves duplicates the results obtained by rotating full skin cuffs about the D-V axis. Skin cuffs rotated for periods from 6 months to over 2 years before amputation are also capable of causing multiple regenerates to form. No significant difference in the percentage of multiple regenerates was seen after skin rotation and limb amputation through shoulder, upper arm, and forearm levels. X-Radiation (4000 r) of either the skin or underlying tissues before skin rotation resulted in single regenerates after amputation. If a strip of normal skin was turned perpendicularly to the long axis of the irradiated underlying stump tissues, the regenerative response was blocked. In some of the above experiments, regenerates with longitudinally duplicated upper arm and forearm segments appeared. It is postulated that normally both the skin and the underlying limb tissues can influence morphogenesis during regeneration and that they work in harmony. In contrast, rotated skin and the underlying tissues each exert a morphogenetic influence upon the regenerating limb, and the regenerate is not able to integrate these disharmonious influences. This is reflected in the highly abnormal morphology of the regenerates. The nature of the morphogenetic influence disrupted by skin rotation is not yet known.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.     

Effects of amiloride analogues on adult Notophthalmus viridescens limb stump currents

We have previously investigated the relevance to limb regeneration of epidermally driven, Na+-dependent limb stump currents by blocking epidermal Na+ channels with amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide. In order to reduce Notophthalmus viridescens stump currents more effectively than with amiloride, we have examined six amiloride analogues. Of these, only benzamil, 3,5-diamino-6-chloro-N-[(benzylamino)aminomethylene]pyrazinecarboxamide, was more effective than amiloride. The concentration of benzamil that reduced stump currents to half their initial value was 0.034 microM, while this concentration for amiloride was 0.42 microM. We also found a contribution of calcium ions to these currents. When immersed in water whose Ca2+ concentration decreased stepwise from 1 to 0 mM, stump currents decreased significantly, although to a variable extent, depending on the batch of newts. With 30 microM benzamil and 0.5 mM calcium (in water that also contained 1.5 mM NaCl and 0.06 mM KCl) stump currents could be reduced to very low levels and, in many cases, changed to incurrents.     

Spondylodiscitis and an aortic aneurysm due to Campylobacter coli

The aim of the current study was to evaluate the prevalence of stump infections after major amputations of the lower extremities. Patients rehospitalized in Hospital de Base of the Medicine School in São José do Rio Preto in the period from January 2005 to January 2007 due to stump infection after major amputations of lower extremities were evaluated in a retrospective study. All the patients underwent prophylactic antibiotic therapy at the time of the surgery. The Fisher exact test was utilized for statistical analysis with an alpha error of 5% (p-value < 0.05) being considered acceptable. A total of 231 patients were submitted to major amputations during this period and 17 (7.3%) were rehospitalized due to amputation stump infections of which 5 (29.4%) died within one month. The association between death due to stump infection and other causes of death during rehospitalizations was not significant (Fisher exact test: p < 0.1). However, death during rehospitalizations was significantly higher than in the initial hospitalization.     

Limb regeneration of the adult newt (Notophthalmus viridescens) in the absence of the spleen

Summary It has been suggested that the immune system might figure prominently in the regulation of forelimb regeneration. However, neither the nature of this influence nor the aspect(s) of regeneration influenced are clearly known. The determination of which components of the immune system are indispensable for regeneration would be a logical first step in attempting to address such questions. This investigation, therefore, examined the effects of removing the spleen, a major lymphoid organ in the newt, upon the progress of regeneration. Splenectomies performed concomitantly with or after forelimb amputation failed to alter the time course of regeneration. Splenectomies, but not sham-splenectomies, performed prior to amputation reduced the time required to achieve successive stages of regeneration under some, but not all conditions, i.e., when performed 10–20 days before amputation, during the late fall and winter. Up until 35 days after amputation, no gross morphological distortions were observed as a result of splenectomy. It was concluded that the spleen is not required for regeneration to occur.Portions of this work constitute part of the thesis submitted by M.E. Fini in partial fulfillment of the requirements for the M.S. degree in Biology at Boston College     

Volumen und Proteinsynthese der Kerne des Stratum Basale in der Stumpfepidermis während des Wundverschlusses nach Amputation der Vorderextremität bei Triturus vulgaris

After injection of tritiated phenylalanine, microautoradiographs of longitudinal sections of the stumps of the forelegs of Triturus vulgairs 1 hour, 2, 3 and 9 hours after amputation and also of the opposite limb (reference) were prepared for quantitative measurements. The epidermal tissue (stratum basale) was examined with regard to proximo-distal changes of nuclear volume and protein synthesis. In the stratum basale of the stump the values for the parameters examined were similar to those of the reference limbs 1h and 2h after amputation, but 3h and 9h after amputation areas of increased nuclear volume were observed as well as an increased rate of protein synthesis per unit of nuclear volume. Protein synthesis in the epidermis of the stump was extremely low 1h after amputation. Protein synthesis in the reference epidermis was also reduced at this time, but then increased although the nuclear volume remained constant.     

The blood vessels of the regenerating limb of the adult newt,Triturus

The vessels of the forelimb stump and regenerate were perfused with Prussian blue and studied as whole mounts and in histological sections to reveal the condition and disposition of the blood vessels in various stages of forelimb regeneration in the adult newt, Triturus viridescens. The development of the vessels in the regenerate seemed to be comparable in all its essential features to that which has been described for the normal developing limb in urodele, chick and pig embryos. The first signs of regeneration of the vessels are seen during wound healing when fine sprouts appear from the old vessels near the amputation wound. These grow and anastomose, but are limited to the transition region between old and new tissues and avoid the growing blastema during the early stages of regeneration. As the regenerate enlarges into a conical structure vessels invade the proximal part of the growth and avoid the distal regions. It is only during the stages of histogenesis and morphogenesis that vessels grow into more distal regions. The regions of most active enlargement of the early or later regenerate are those most poorly vascularized. These results are discussed against the background of the activity of certain enzymes during regeneration. In the advanced regenerate, preferential channels are consolidated until in the palette and digital stages the pattern of the blood vessels resembles that of the normal limb.